Compared to DNA, molecular recognition of double stranded RNA has received relatively little attention. Until the early 90's, RNA was viewed as a passive messenger in the transfer of genetic information from DNA to proteins. However, since the discovery that RNA can catalyze chemical reactions, the number and variety of non-coding RNAs and the important roles they play in biology have been growing steadily.[1] Currently, the functional importance of most RNA transcripts is still unknown and likely that many more regulatory RNAs will be discovered in the near future. The ability to selectively recognize and control the function of such RNAs will be highly useful for both fundamental research and practical applications. However, recognition of double helical RNA by sequence selective ligand binding is a formidable challenge.[2, 3]
Double-helical RNA has become an attractive target for molecular recognition because many noncoding RNAs play important roles in the control of gene expression. Recently, short peptide nucleic acids (PNA) were found to bind strongly and sequence selectively to a homopurine tract of double-helical RNA via formation of a triple helix. Molecular recognition of RNA can be enhanced by α-guanidine modification of PNA. Ly and coworkers noted that guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple-helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double-helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from D-arginine recognized the transactivation response element of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNA and the purine-rich strand of the bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex noncoding RNAs.
Biologically relevant double helical RNAs may be recognized by major groove triple helix formation using peptide nucleic acid (PNA).[4-6] PNAs as short as hexamers form stable and sequence selective Hoogsteen triple helices with RNA duplexes (Ka>107 M−1) at pH 5.5.[4] A limitation of triple helical recognition was the requirement for long homopurine tracts, as only the Hoogsteen T(U)*A-T(U) and C+*GC triplets could be used (FIG. 1). Modification of PNA with 2-pyrimidinone[7] and 3-oxo-2,3-dihydropyridazine (E)[8] nucleobases allowed efficient and selective recognition of isolated C-G and U-A inversions, respectively, in polypurine tracts of double helical RNA at pH 6.25.[5, 6] However, the high affinity of PNA at pH 5.5 was greatly reduced at pH 6.25 and no binding could be observed at physiologically relevant salt and pH 7.4.[5] The remaining problem was the unfavorable protonation of cytosine, which was required for formation of the Hoogsteen C+*G-C triplets (FIG. 1). Because its pKa=4.5, cytosine is hardly protonated under physiological pH, which greatly decreases the stability of the triple helix.
Povsic and Dervan pioneered the chemical modulation of the cytosine pKa by showing that triple helices containing 5-methylcytosine were more stable at higher pH than those of unmodified DNA.[10] More recently, derivatives of 2-aminopyridine have been used to increase the stability of DNA triple helices at high pH. [11-14]
An alternative approach has used neutral nucleobases that mimic the hydrogen-bonding scheme of protonated cytosine. The most notable examples are pseudoisocytosine (abbreviated as J in FIG. 1) by Kan and co-workers,[15] methyloxocytosine by McLaughlin and co-workers,[16, 17] and a pyrazine derivative by von Krosigk and Benner.[18] The J base is widely used in PNA to alleviate the pH dependency of PNA-DNA triplexes.[19, 20]
Practical applications of triple-helical recognition of nucleic acids are limited by (1) the low stability and slow formation of the triplex caused, at least in part, by electrostatic repulsion between the negatively charged phosphate backbones of the double helix and the incoming third-strand oligonucleotide and (2) the requirement for long homopurine tracts, as only U*A-U and C*G-C triplets are used in the common triple-helical recognition. However, it was recently shown that short peptide nucleic acids (PNA) recognize double-helical RNA via highly stable and sequence selective triple-helix formation.[4-5] PNA, as short as hexamers, formed triple helices with a RNA duplex faster and with higher affinity than with RNA as the third strand. Furthermore, nucleobase modifications allowed recognition of isolated pyrimidine inversions in short polypurine tracts, thus expanding the potential of recognition to biologically relevant double-helical RNA, such as rRNA and microRNAs. [5]